Fig 2: Reduced FOXO1 reexpression in old individuals favors differentiation into granzyme B–expressing effector cells and promotes secretion of B cell cytotoxic exosomes.(A and B) Naïve CD4+ T cells from young healthy individuals were activated with anti-CD3/anti-CD28 beads for 3 days followed by 2-day culture with vehicle or 50 nM FOXO1 inhibitor (AS1842856). Representative histograms (left) of intracellular granzyme B after restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (A). The shaded histogram represents isotype control. Data (right) from six young healthy individuals (two-tailed paired t test). Expression of GZMB, PRF1, PRDM1, and RUNX3 determined by RT-PCR (B). Results are presented relative to vehicle-treated cells (two-tailed paired t test). (C) Naïve CD4+ T cells were activated with anti-CD3/anti-CD28 beads for 5 days. Representative histograms (left) of intracellular granzyme B after restimulation with PMA and ionomycin and cumulative data (right) in cells from seven 20- to 35-year-old and seven 65- to 85-year-old healthy individuals (two-tailed unpaired t test). (D and E) Unstimulated naïve CD4+ T cells from six 65- to 85-year-old healthy individuals were transfected with a TFEB expression vector or a control vector and then stimulated with anti-CD3/CD28 beads for 5 days. Representative histograms (left) of and cumulative data (right) of intracellular granzyme B after restimulation with PMA and ionomycin (two-tailed paired t test) (D). The shaded histogram represents isotype control. Gene expression was measured by RT-PCR (E). Results are expressed relative to control vector–transfected cells (n = 6; two-tailed paired t test). **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Naïve CD4+ T cells from young individuals were cultured for the past 24 hours of the 5-day culture in exosome-depleted medium with vehicle or 50 nM FOXO1 inhibitor (AS1842856). Exosomes were isolated and assessed for granzyme B by immunoblotting. Western blots representative of three experiments. (G and H) Freshly isolated peripheral blood B cells were treated with exosomes isolated as in (F) for 24 hours. Caspase-3 activity in B cells was determined by immunoblotting using an antibody recognizing both uncleaved and cleaved caspase-3. Western blots representative of two experiments (G). Relative frequencies of Annexin V+ 7-AAD+ B cells (H); n = 3, two-tailed paired t test. (I and J) Exosomes from young and old naïve CD4+ T cells were isolated as in (F). Exosomal granzyme B was detected by immunoblotting. Representative Western blots of cells from three young and three old individuals (I) and frequencies of apoptotic B cells induced by exosomes isolated from four young and four old individuals (J); two-tailed unpaired t test, **P < 0.01.

Fig 3: The influence of LINC02474 on CRC cells can be mediated by restricting transcriptional regulation of GZMB. (A) qRT-PCR and (B) Western blotting analysis for the expression of GZMB by siRNAs. (C–F) Migration and invasion after GZMB depletion in DLD-1 and LOVO cells, in which LINC02474 was stably depleted. Representative images (original magnification, ×100) are shown. (G, H) Expressions of apoptosis-related proteins and GZMB after depleting GZMB in DLD-1 cells, in which LINC02474 was down-regulated. (I) Dual-luciferase reporter gene vector of GZMB gene promoter. (J) Relative luciferase activity of GZMB gene promoter after LINC02474 was depleted. Results are means ± SD. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001.

Fig 4: Pathogenic effector CTLs enriched in PWPs compared to NSPs. a t-SNE plot of CD8B, CD103, CD69, and PDCD1 expression levels on CTLs. b Bubble plots showing significant ligand/receptor pairs between CTLs and other cell types. Point sizes indicate permutation p values (CellPhoneDB). c IF staining showing the infiltration of CD3+ CD8+ GZMB+ CTLs in PWPs. 5× magnification (right). Scale bars, 100 μm. d Volcano plots of differentially expressed genes in CTLs obtained from PWP relative to NSP. Hurdle model likelihood ratio test for differential expression, Benjamini–Hochberg multiple testing correction. All genes are given in Additional file 18: Table S9. e IF staining showing the co-expression of PRF and GZMB in CTLs. Scale bars, 100 μm. f Cell densities (number of cells/villus) of CD3+ CD8+ GZMB+ CTLs and GZMB+ PRF+ CTLs of NSPs and PWPs, according to Fig. 6c and e. ***p<0.001. Student’s t-test. Error bars: SEM (n=6). g Violin plot showing the expression of costimulatory and exhausted gene sets in CTLs of NSPs and PWPs. Wilcoxon test. *p<0.05, **p<0.01, ***p<0.001

Fig 5: CGA up regulated Granzyme B (GZMB) expression in tumor tissues. The tumor tissues of the anti-PD-1 antibody monotherapy group (NS+Anti-PD-1, n = 3) and the CGA + anti-PD-1 antibody combination treatment group (CGA+Anti-PD-1, n = 3) from the 4T1 tumor bearing mice were collected. Total RNA of tumor tissues was extracted and the RNA sequence analysis was performed using “limma” and “DESeq” R packages. The differential expression genes (DEGs) were enriched in GO functions by DAVID database. A. DEGs comparison was shown in volcano plot with |log2FC| ≥ 1 and p-Value ≤ 0.05. B. The top 20 enrichment of GO biological process function terms. C. Heatmap of gene expression for cytolysis function of the NS+Anti-PD-1 and CGA+Anti-PD-1 group. Scale represents log transformed TPMs + 1, the red to blue represents the relatively gene expression from high to low. In the experiment groups, NS+Anti-PD-1-1, NS+Anti-PD-1-2, NS+Anti-PD-1-3, and CGA+Anti-PD-1-1, CGA+Anti-PD-1-2, CGA+Anti-PD-1-3 are the samples that were sequenced. D-E. CGA+Anti-PD-1 treatment up regulated the protein expression of GZMB in tumor tissues. Representative multi color immunofluorescent images stained of CD8 (pink) and GZMB (green) in tumor tissues (D). Scale bar. 10 μm. The protein expression of GZMB was tested by Western blot analysis (E). The results of GZMB were normalized to β-actin, as density ratio. Data are presented as mean ± SD (n = 6). Significant differences are indicated: *p< 0.05, **p< 0.01, vs NS+Anti-PD-1 group by Student's t-test. GZMB indicated granzyme B.